Subcloning, Expression and Purification of Functional E. Coli Nucleotide Excision Repair Protein Uvra Using IMPACT-CN System Cathy W

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5-2014 Subcloning, Expression and Purification of Functional E. coli Nucleotide Excision Repair Protein UvrA Using IMPACT-CN System Cathy W. Lin Mrs East Tennessee State University

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______

A thesis

presented to

the faculty of the Department of the Biology

East Tennessee State University

In partial fulfillment

of the requirements for the degree

Master of Science in Biology

______

Cathy W. Lin

May 2014

______

Yue Zou, Chair, PhD

Phillip Musich, PhD

Cerrone Foster, PhD

Keywords: E. coli Nucleotide Excision Repair, UvrABC Nuclease System, IMPACT-CN Protein Purification System ABSTRACT

Subcloning, Expression and Purification of Functional E. coli Nucleotide Excision Repair

Protein UvrA Using IMPACT-CN System

Cathy Lin

DNA in cells is constantly damaged by both endogenous and exogenous genotoxic agents.

Nucleotide excision repair (NER) in Escherichia coli (E. coli) is one of the DNA repair systems that recognizes and removes a variety of DNA damage such as pyrimidine dimers, bulky chemical adducts, DNA intrastrand cross-links, etc. The genes responsible for E. coli NER incisions are UvrA, UvrB, and UvrC. Purification of UvrA, UvrB, and UvrC is essential for research to understand the molecular mechanisms of NER and carcinogenesis. Although UvrA has been successfully purified in our lab in the past, the experimental procedures were very time- consuming and technically challenging. In this study we employed IMPACT (Intein Mediated

Purification with an Affinity Chitin-binding Tag) system to subclone the cDNA of UvrA and express and purify the recombinant UvrA protein by a single-column step. The purified UvrA protein was found to be fully functional in the UvrABC incision assay.

DEDICATION

This research work is dedicated to my beloved and my family for their support, advice, and encouragement.

ACKNOWLEDGEMENTS

I would like to thank my graduate committee for their guidance and mentorship in my graduate study. Thank you Dr. Yue Zou for chairing my committee and for your advice. Through this experience I have gained invaluable knowledge under your help and support. I would want to thank Dr. Phillip Musich and Dr. Cerrone Foster for your advice, encouragement, knowledge, and assistance with my research. I have acquired valuable skills under the guidance of all of you and I will be able to attain my professional goals because of you.

I would also like to thank my fellow graduate students in our lab for their constant help with my research and friendship. Finally, I would like to thank my family and in particular my husband for the massive amount of support and energy they put forward to help me accomplish this degree.

TABLE OF CONTENTS Page

ABSTRACT ...... 2

DEDICATION ...... 3

ACKNOWLEDGEMENTS ...... 4 LIST OF FIGURES ...... 7

Chapter

1. INTRODUCTION ...... 8

2. MATERIALS AND METHODS ...... 16

Subcloning of UvrA Gene into pTYB2 Expression Vector...... 16

Overexpression of UvrA Protein ...... 17 Purification of UvrA Protein ...... 17

UvrA Protein Concentration ...... 18

UvrABC Incision Assays ...... 19

3. RESULTS ...... 20

Obtaining UvrA Gene from the Plasmid pSST10 Carrying UvrA ...... 20

Preparing the C-terminal Fusion Vector pTYB2 For UvrA Gene Insertion ...... 21

Ligation and Transformation ...... 23

Plasmid Isolation from AmpR Transformants ...... 24

Optimizing the Time Course of Induction of UvrA Overexpression by IPTG...... 27

Small Scale UvrA Purification by Chitin Beads to Check DTT Cleavage Efficiency 28

Cell Lysis Efficiency Comparison Between Sonication and French Press...... 31

Purification of UvrA Protein from One Liter of Culture by T7 IMPACT System .... 33

UvrABC Incision Assay to Characterize the Purified UvrA Protein in vitro ...... 36

Chapter Page

4. DISCUSSION ...... 37 REFERENCES ...... 41

VITA ...... 46

LIST OF FIGURES

Figure Page

1. Linear representation of the gene UvrA………………………………………………… 10

2. Prokaryotic nucleotide excision repair ………………………………………………… 11

3. Schematic illustration of IMPACT system ….………………………………………… 13

4. pTYB2 Vector ……..…………………………………………………………………....14

5. The multiple cloning sites of pTYB2 vector ...………………………………………….14

6. Analyzing the PCR reaction products of UvrA gene ……………………………...... 20

7. Confirmation of amplified pTYB2 with various restriction endonucleases……...... 22

8. The purification of UvrA and vector pTYB2 fragments for the ligation…………...... 23

9. PCR products using isolated plasmids as the templates.………………….………...... 25

10. Double-digestion of isolated plasmids (from colonies A, B, and C) by restriction

enzymes NdeI & XhoI ………………………………………………………………..... 25

11. Confirmation of the amplified construct of pTYB2_UvrA with restriction enzymes

Nde I & Xho I.………………………………………………………………………...... 27

12. Optimization of the time course for the overexpression of UvrA protein ………...... 28

13. DTT cleavage efficiency for the binding between the fusion protein and chitin beads ...30

14. Comparison of cells lysis effects between the methods of sonication and French press..32

15. Expression and purification of the UvrA protein using the IMPACT system …………. 34

16. Comparison among the fractions of the eluted target protein UvrA .………………..…. 35

17. Purified protein UvrA samples from pool-1 and pool- 2……………………………….. 35

18. UvrABC incision assay…………………………………………………………………. 36

CHAPTER 1

INTRODUCTION

DNA in cells is under constant attack from both endogenous and exogenous genotoxic agents (Zou et al. 1995; Zou et al. 2004; Young et al. 2005; Liu 2007). Exogenous sources could be manmade mutagenic substances and naturally occurring agents such as sunlight and dietary mutagens. Reactive oxygen species formed during cellular metabolism could be one of the endogenous sources. According to the type of DNA structural alterations, the DNA damage can be classified as damage to the nucleotide base, damage to the phosphodiester backbone, and

DNA cross-links. The results can lead to permanent changes in the genetic information encoded in the DNA due to its mutations. The DNA damages are physical and chemical abnormalities in the DNA molecule. The DNA repair of a cell is vital to the integrity of its genome and thus to the normal functionality of that organism. In response to this threat, cells detect and repair the DNA damage.

A number of DNA repair systems have evolved and each of them has developed to specialize in the repair of certain types of damage. Nucleotide excision repair (NER) is a major repair pathway that is known for removal of stretches of bases containing various lesions such as pyrimidine dimers, bulky chemical adducts, DNA intrastrand crosslinks, and some forms of oxidative damages. The common features of these lesions are DNA duplex helical distortion and

DNA chemical modification (Zou et al. 1995; Zou et al. 2001; Zou et al. 2003; Yang et al. 2005;

Zou et al. 2005). Soon after the first detection of NER in 1960 when the excision of UV-induced

DNA lesions in bacteria was observed (Setlow and Carrier 1964; Boyce and Howard-Flanders

1964), following studies revealed the genes responsible for bacterial NER incisions are UvrA,

UvrB, and UvrC (Hanawalt and Haynes 1965).

The NER pathway in Escherichia coli also involves the UvrABC proteins. The UvrA,

UvrB, and UvrC proteins recognize and incise damaged DNA in a multistep reaction (Figure 2).

In solution, UvrA dimer formation is driven by ATP (Mazur and Grossman 1991). UvrA forms either an UvrA2B (Orren and Sancar 1989) or UvrA2B2 (Verhoeven et al. 2002) complex with

UvrB. UvrA initiates the DNA contacts and transfers the DNA to the DNA binding domain of

UvrB (Della Vecchia et al. 2004). UvrB is considered the central recognition protein in the bacterial NER system (Sancar et al. 1988; Orren et al. 1992), and its cryptic ATPase activated in the presence of the UvrAB:DNA complex is necessary for damage verification. The interaction of UvrA2B with the damage causes unwinding, denaturing, and opening of the local DNA duplex at the adduct (Zou and Van Houten 1999). UvrA hydrolyzes ATP, resulting in its self- dissociation from the recognition complex and leaving behind a UvrB:DNA preincision complex

(Orren and Sancar 1990). UvrB must be in its ATPase-bound conformation (Moolenaar et al.

2000) before 3’ incision by UvrC, which is responsible for both the 3’ and 5’ incision reactions

(Verhoeven et al. 2000). The UvrBC complex is a structure-specific, ATP-dependent endonuclease (Zou et al. 1996). The first incision is at the fourth phosphodiester bond 3’ of the lesion, and the second incision is at the eighth phosphodiester bond 5’ of the damaged base

(Sancar and Rupp 1983; Lin and Sancar 1992; Lin et al. 1992; Zou et al. 1995; Verhoeven et al.

2000). Following incision, UvrC dissociates and DNA helicase II (UvrD) is required to release the incised oligonucleotide containing the lesion (Sancar and Rupp 1983; Lin and Sancar 1992;

Lin et al. 1992; Verhoeven et al. 2000). DNA polymerase I fills this gap and removes UvrB from the nondamaged DNA stand (Caron et al. 1985; Hasain et al. 1985). DNA ligase I joins the

9 newly synthesized end to the parental DNA, thus completing the NER pathway. Overall, UvrA plays a vitally important role in the NER mechanism because it is the first component of the system to recognize DNA damage. The UvrA gene encodes a 115 kDa protein. Sequence analysis has revealed the presence of 2 zinc fingers and 2 ATP-binding cassette ATPase domains. UvrA (Figure 1) consists of 2 halves (white and yellow, respectively) separated by a flexible protease sensitive linker region.

Figure 1: Linear representation of the gene UvrA. Two domains are separated by a flexible protease-sensitive linker region. Within each domain, there is one ABC ATPase motif, a zinc finger. The 2 domains are shown in gray and yellow, while the linker region is shown in beige. The conserved ABC ATPase motifs are shown in red and orange and the zinc fingers are shown in blue and green.

It is likely that the C-terminal zinc finger is primarily responsible for UvrA’s DNA binding capacity that facilitates the NER reaction (Houten et al. 2005; Truglio et al. 2006). N-terminal domain possessed both the ability to dimerize and hydrolyze ATP. The dimerization of UvrA is in a head-to-head fashion (Myles and Sancar 1991), and it could be a key regulatory point in the

NER pathway. UvrA is a DNA-independent ATPase that can hydrolyze both ATP and GTP

(Seeberg and Steinum 1982; Caron and Grossman 1988; Truglio et al. 2004).

Figure 2: Prokaryotic nucleotide excision repair (Shell 2008)

Protein UvrA, UvrB, and UvrC are required for the bacterial NER research study in our lab.

Although the protein UvrA has been successfully purified in the past, the experimental procedures were very time-consuming and technically challenging. In this study we employed

IMPACT (Intein Mediated Purification with an Affinity Chitin-binding Tag) system, a single- column step method, to subclone the gene of UvrA, and express and purify the recombinant

UvrA protein. The IMPACT-CN system (Figure 3) is an intein-based affinity protein purification system. Intein, encoded from the Saccharomyces cerevisiae VMA1 gene, is a protein splicing element with 454 amino acid residues. In this system there is an inducible (controllable) peptide bond cleavage reaction involved either at the N-terminus or C-terminus of an intein. To release the target protein from the fusion protein, the peptide bond cleavage is triggered or induced by addition of thiols such as dithiothreitol (DTT), β-mercaptoethanol or free cysteine at either N- terminus or C-terminus of an intein. Specific mutations at the C-terminal splice junction of the intein allow C-terminal peptide bond cleavage induced by addition of thiols such as DTT, resulting in elution of the target protein while leaving the intein tag remains on the affinity column (NE BioLabs Inc. manual Page 3).

The pTYB vectors contain a T7/lac promoter to provide strict control of the fusion gene expression. These vectors use their own copy of the lac I gene encoding the lac repressor.

Binding of the lac repressor to the lac operator suppresses basal expression of the fusion gene in the absence of IPTG (isopropyl β-D-1-thiogalactopyranoside) induction. The pTYB vectors also carry the bla gene to encode ampicillin selective marker, which conveys ampicillin resistance to the host strain. The pTYB2 vector is one of the examples, and it uses a T7/lac promoter and carries its own copy of the lac I gene and bla gene (NE BioLabs Inc. manual Page 7).

Figure 3: Schematic illustration of IMPACT system (NE BioLabs Inc. manual Page 3)

Figure 4: pTYB2 vector

Figure 5: The multiple cloning sites of pTYB2 vector (NE BioLabs Inc. manual page 8)

In this project we chose the C-terminal fusion vector pTYB2 (7,474 bp; Figure 4), which allows the fusion of the cleavable intein tag to the C-terminus of the target protein UvrA, for cloning and expression of recombinant protein UvrA in E. coli cells. pTYB2 uses ATG of the

Nde I site in the multiple cloning region (Figure 5) for translation initiation. Use of the Xho I site pTYB2 yields the target protein UvrA with 3 (glycine, proline, and glutamate) extra residues at the C-terminus of target protein UvrA after the cleavage of the self-cleavable intein tag. The chitin binding domain (CBD) in the intein tag allows the binding of the fusion precursor protein

UvrA_intein to a chitin column. The intein tag undergoes specific self-cleavage in the presence of thiols, such as DTT, β-mercaptoethanol, or free cysteine and releases the target protein UvrA from itself resulting in a single-column purification of the target protein UvrA.

CHAPTER 2

MATERIALS AND METHODS

Subcloning of UvrA Gene into pTYB2 Expression Vector

For subcloning of the UvrA gene from plasmid pSST10 carrying UvrA (supplied by L.

Grossman, Johns Hopkins University), two DNA oligodeoxyribonucleotides, 5’ sense primer, containing a NdeI-restriction site GGG AAT TCC ATA TGA TGG ATA AGA TCG AAG TTC

GGG, and 3’ antisense primer, containing a XhoI restriction site CCG CTC GAG CAG CAT

CGG CTT GAG GAA G were synthesized. The UvrA gene was amplified by polymerase chain reaction (PCR) in a 50 microliter (µl)-reaction mixture containing 1 x LongAmp Taq reaction buffer (60 mM Tris-SO4, 20 mM (NH4)2SO4, 2 mM MgSO4, 3% Glycerol, 0.06% IGEPAL®

CA-630, 0.05% Tween® 20, pH 9 @ 25°C), 300 micromolar (µM) deoxynucleotide triphosphates (dNTPs), 400 picomolar (pmol) of each primer, 30 nanogram (ng) template DNA

(pSST10_UvrA) and 2 units (2 µl) LongAmp Taq DNA polymerase using following conditions:

I cycle (initial denaturation): 940C for 30 seconds; 30 cycles: 940C for 30 seconds, 580C for 60 seconds, 650C for 3 minutes, followed by 650C for 10 minutes. The PCR product was purified using QIAquick PCR purification kit available from Qiagen (Cat. 28104). Purified PCR product as well as vector pTYB2 were double-digested with restriction endonucleases NdeI + XhoI, vector pTYB2 was dephosphorylated with alkaline phosphatase (1 unit of FastAP, Thermo

Scientific, #EF0651). Both DNA fragments were purified from 1% agarose gel following gel electrophoresis and extracted by QIAquick Gel Extraction kit (Qiagen, cat.28704). Purified DNA

16 fragments of UvrA and vector pTYB2 were used (molar ratio 3:1) in ligation reaction by 1 unit of T4 DNA ligase (Promega, M1801) at 40C overnight.

Ligation mixture was transformed into E. coli DH5α competent cells. The plasmids from

AmpR transformants were isolated, analyzed by digestion with both Nde I and Xho I restriction endonucleases, and sequenced to confirm that both junctions’ sequences between UvrA DNA fragment and the pTYB2 vector were correct.

Overexpression of UvrA Protein

The resulting recombinant plasmid DNA, pTYB2-UvrA, was transformed into E. coli

C41 (DE3) cell, a derivative of BL21 (DE3). The fresh AmpR transformants were used to inoculate 10 ml lysogeny broth (LB) medium, with final concentration 100 µg/ml ampicillin, for

1 hour at 370C first, then this culture was transferred into 1 liter of LB medium (with 100 µg/ml ampicillin) and grew for 2.5 hours at 370C with shaking (200 rpm) until the optical density at 600 nm (OD600) of the culture was around 0.6, then 0.7 mM IPTG was added. The IPTG induction of

UvrA was at 300C for 6 hours with 200 rpm shaking. The culture was harvested by 6,000 rpm centrifugation and cell pellets were stored at -200C overnight.

Purification of UvrA Protein

The pellets from 1 liter of culture were resuspended in 80 ml of column buffer (20 mM

Tris-HCl, pH 8.0; 500 mM NaCl; 0.1 mM EDTA; 0.1% Triton X-100) containing 1 mM phenylmethylsulfonyl fluoride (PMSF). Cells were lysed by 1 pass through French press at 8,000 psi, and clarified by centrifugation at 13,200 rpm for 30 minutes at 40C. The clarified extracts were used for loading the chitin column (10 ml) at a rate of no faster than 0.5 ml/min after chitin beads were equilibrated with 15 volumes of column buffer. The column then was washed with

30 column volumes of column buffer at 1 ml/min flow rate. For inducing the on-column cleavage reaction of the fusion protein UvrA-intein, the column was flushed quickly with 3 column volumes of cleavage buffer [20 mM Tris-HCl, pH 8.0; 500 mM NaCl; 0.1 mM EDTA; freshly diluted 30 mM dithiothreitol (DTT)]. The flow in the column was stopped and the cleavage was allowed to continue at 40C overnight. Protein UvrA was eluted using additional 3 column volumes of cleavage buffer without DTT, and was collected in 1 milliliter fractions for total of 20 milliliters. Fraction 2 and fraction 4 were examined using PAGE for molecular weight and purity of isolated protein. Samples from some major steps of overproduction and purification of UvrA protein were separated on a 10% SDS-polyacrylamide gel that was stained with

Coomassie blue and photographed. Such samples included that from cell extracts of noninduced cultures, IPTG-induced cell cultures, the flow-through of extract loading, column washing, and on-column cleavage induction, chitin resin, and elution fractions 2 and 4, respectively. Fractions

1-10 (pool-1) and 11-20 (pool-2) were pooled and dialyzed against one liter of storage buffer (50 mM Tris-HCl; 100 mM KCl; 0.1 mM EDTA; 0.1 mM DTT; 50% glycerol). The obtained UvrA protein samples were aliquoted into several microcentrifuge tubes and stored at -200C until the biological activity was confirmed.

UvrA Protein Concentration

The concentrations of UvrA protein samples were determined by the microplate procedure (Thermo Scientific Pierce 660nm Protein Assay) with bovine serum albumin (BSA) as a standard. A standard curve was prepared within the assay’s working range. 10μL of each replicate of standards [800 µg/mL, 400 µg/mL, 200 µg/mL, 100 µg/mL, and double-distilled

(dd)-H2O], UvrA protein samples (1:10 dilution with dd-H2O), and the blank samples (storage buffer in 1:10 dilution with dd-H2O) were added into the microplate well of 96-well plates. Then

150 μL of the protein assay reagent was added into each well. The plate was covered and mixed on a plate shaker at medium speed for 1 minute, and incubated at room temperature for 5 minutes. The blank wells were used to zero the plate reader first, and the absorbance of the standards and UvrA protein samples were measured at 660 nm. A standard curve was prepared by plotting the average blank-corrected 660 nm measurement for each BSA standard versus its concentration in μg/mL, and this standard curve was used to determine the concentrations of

UvrA protein samples from pool-1 and pool-2. The concentrations of pool-1 and pool-2 are 2.0

µM and 1.3 µM, respectively.

UvrABC Incision Assays

The 5’ termini 32P-labeled DNA substrates (4 nM) containing FABP [N-(20- deoxyguanosin-8-yl)-4-ﬂuoro-4-aminobiphenyl] adduct were incised by UvrABC (UvrA, 20 nM;

UvrB, 250 nM; UvrC, 100 nM) in UvrABC buffer (50 mM Tris-HCl, pH 7.5, 50 mM KCl, 10 mM MgCl2, 5 mM DTT, 1 mM ATP) at 370C for a specified length of time. UvrABC enzymes were diluted and premixed in Uvr storage buffer (20 mMTris-HCl, 500 mM NaCl, 1 mM EDTA,

50% glycerol) prior to addition to incision reaction mixes. Aliquots were collected at 0, 10, 20, and 30 minutes into the reaction. The reaction was terminated by heating at 950C for 5 minutes.

The products were denatured into single strands by addition of formamide 6 x dye (bromophenol blue and xylene cyanol) loading buffer and heating to 950C for 5 minutes, and immediately plunged into ice to prevent reannealing. Digested (incision) products then were analyzed by gel electrophoresis on a 12% polyacrylamide sequencing gel under denaturing conditions with TBE buffer (50 mM Tris-borate, 1 mM EDTA pH 8.0). 32P-labeled incision products or nonincision

DNA bands in the gels were visualized using a Fuji FLA-5000 image scanner with MultiGauge

V3.0 software.

CHAPTER 3

RESULTS

Obtaining UvrA Gene from the Plasmid pSST10 Carrying UvrA

The UvrA gene was amplified from the plasmid pSST10 carrying UvrA by PCR

(Polymerase Chain Reaction) before it could be cloned into the expression vector pTYB2 (7474 bp) using the IMPACTTM kit (NEB). The first step was the design of the forward (5’ sense) and reverse (3’ antisense) primers: forward primer containing a NdeI-restriction site GGG AAT

TCC ATA TGA TGG ATA AGA TCG AAG TTC GGG, and reverse primer, containing a XhoI restriction site CCG CTC GAG CAG CAT CGG CTT GAG GAA G were synthesized. The PCR products were purified and double-digested with restriction enzymes NdeI & XhoI, and were analyzed by 1% agarose gel electrophoresis (UvrA gene has 2,823 bp) as indicated in Figure 6.

1 2 3

10,000 bp 8,000 6,000 5,000

4,000 3,500 3,000 2,500 2,000

1,500

Figure 6: Analyzing the PCR Reaction Products of UvrA Gene. Products of PCR reaction using pSST10_UvrA plasmid as the template as the positive control (Lane 1). Products of PCR reaction using dd-H2O as the template as the negative control (Lane 2). Purified PCR products were double-digested with restriction enzymes NdeI and XhoI (Lane 3).

Preparing the C-terminal Fusion Vector pTYB2 For UvrA Gene Insertion

The vector pTYB2 purchased from New England Biolabs was first amplified by transformation into DH5α competent cells and plasmid isolation. The newly amplified plasmid of pTYB2 vector then was confirmed by the digestion with various restriction endonucleases and analyzed by 1% agarose gel electrophoresis (Figure 7). Meanwhile, the efficiency of restriction endonucleases Xho I and Nde I was affirmed.

A 1 2 3 4 5

10,000 bp 8,000

6,000 5,000 5242

4,000 3,500 3,000 2,500 2232 2,000

I I B I I EcoR EcoR coRI/BglI oRI/Bgl I XhoI XhoI/NdeIE Ec

10,000 bp 8,000

6000 5,000 4,000 3,500 3,000 5 Prime 5 Prime

Figure 7: Confirmation of amplified pTYB2 with various restriction endonucleases. (A) DNA Marker (GeneRuler 1 kb DNA Ladder, Thermo Scientific #SM0313) (Lane 1). Circular pTYB2 plasmid (non-cutter) (Lane 2). pTYB2 digested with restriction endonuclease Xho I (single cutter) (Lane 3). pTYB2 digested with restriction endonuclease Nde I (single cutter) (Lane 4). pTYB2 digested with restriction endonuclease Bgl I (dual cutter) (Lane 5). (B) pTYB2 digested with single cutters Xho I, EcoR I, Xho I/Nde I, EcoR I/Bgl I, respectively. QIAprep Spin Miniprep kit (Cat. 27104) were used for the plasmid isolation and purification except 2 samples were purified by PerfectPure DNA Kit (by 5 Prime, Cat. 2900289) as indicated in the figure.

Ligation and Transformation

Purified PCR products as well as vector pTYB2 were double-digested with restriction

endonucleases Nde I and Xho I. The vector pTYB2 was further dephosphorylated with alkaline

phosphatase (FastAP). Both DNA fragments were purified from 1% agarose gel electrophoresis

(Figure 8) using QIAquick Gel Extraction kit. The bands showing in lane 1 and 2 are double-

digested vector pTYB2 and UvrA, respectively.

1 2

10,000 bp 8,000

6,000 5,000

4,000 3,500 3,000 2,500 2,000

Figure 8: The purification of UvrA and vector pTYB2 fragments for the ligation. Vector pTYB2 was double-digested with restriction endonucleases NdeI and XhoI, and then dephosphorylated with FastAP (alkaline phosphatase) (Lane 1). PCR products (UvrA fragment) were double- digested with restriction endonucleases NdeI and XhoI (Lane 2). Both of DNA fragments were purified from 1% agarose gel electrophoresis using QIAquick Gel Extraction kit.

The UvrA DNA fragment and the prepared vector pTYB2 were combined into a reaction with the enzyme T4 DNA ligase at 40C overnight, which covalently links free ends of DNA together. This ligation reaction mixture was transformed into E. coli DH5α competent cells under heat shock, which allows putting ligated pTYB2_UvrA DNA plasmid into the bacterium. The transformants then were plated onto LB-ampicillin agar media, which allowed only cells that had successfully taken up ligated pTYB2_UvrA DNA plasmid to grow. After the incubation at 370C overnight, there were over 20 colonies found growing on the LB-ampicillin agar media plate.

Plasmid Isolation from AmpR Transformants

Isolated plasmids named 1 through 6, and A, B, and C were obtained from 2 different ligation reactions and transformations, respectively. The purified plasmids from all 9 of these colonies were used as the templates for the PCR reactions by the pair of primers for UvrA gene described in Experimental Procedures, and the PCR products were analyzed by 1% agarose gel

(Figure 9). Very likely, all these plasmids isolated, including colony B, harbored UvrA gene

(2,823 bp).

Isolated Plasmids C( olonies)

+ - 1 2 3 4 5 6 A B C

10,000 bp 8,000

6,000 5,000 4,000 3,500 3,000 2,500

2,000

Figure 9: PCR Products using isolated plasmids as the templates. PCR products using pSST10_UvrA plasmid as the template (positive control). PCR products using pcDNA3.1(+)_ATR plasmid as the template (negative control). Isolated plasmids named 1 through 6, and A, B, and C were obtained from 2 different ligation reactions and transformations, respectively.

1 2 3 4 5

10,000 bp 8,000

6,000

5,000

4,000

Figure 10. Double-digestion of isolated plasmids (from colonies A, B, and C) by restriction enzymes NdeI & XhoI. The double-digestion reaction was conducted as described in Experimental Procedures. Untreated circular vector pTYB2 (lane 1), double-digested vector pTYB2 (lane 2), double-digested plasmid from colony A (lane3), colony B (lane 4), colony C (lane 5). All 3 colonies were double-digested by NdeI and XhoI restriction enzymes for 3 hours.

All three of the purified plasmids from colony A, B, and C were double-digested by restriction endonucleases NdeI and XhoI at 370C for 3 hours and then were analyzed by 1% agarose gel electrophoresis (Figure 10) showing that colony B could be the pTYB2_UvrA construct.

The possible construct of pTYB2_UvrA (Colony B) was further amplified by transformation into E. coli DH5α competent cells. The plasmids from AMPR transformants

(colonies B1 and B2) were isolated, analyzed by double-digestion with restriction endonucleases

Nde I and Xho I, and analyzed by 1% agarose gel electrophoresis (Figure 11). The plasmids from both transformants (colonies B1 and B2), the same as their original plasmid purified from colony

B, were all very likely the construct of pTYB2_UvrA.

The final construct of pTYB2_UvrA was confirmed by DNA sequencing that revealed that both C and N-terminal junctions’ sequences between inserted UvrA fragment and the prepared vector pTYB2 were correct. The plasmid of pTYB2_UvrA construct was transformed into C41 (DH3) E. coli competent cells for protein overexpression.

1 2 3 4 5 6

10,000 bp 8,000

6000 5,000 4,000 3,500 3,000

2,500

2,000

Figure 11: Confirmation of the amplified construct of pTYB2_UvrA with restriction enzymes Nde I & Xho I. DNA ladder (Lane 1). Circular vector pTYB2 plasmid (Lane 2). Double-digested vector pTYB2 with NdeI & XhoI (Lane 3). Double-digested plasmid (from colony B1) with Nde I & Xho I (Lane 4). Double-digested plasmid (from colony B2) with Nde I & Xho I (Lane 5). Protein UvrA from pool-32B (Lane 6).

Optimizing the Time Course of Induction of UvrA Overexpression by IPTG

In order to determine the time course of induction, 120 ml culture in the flask was grown

0 at 37 C in LB medium until the OD600 reached 0.6 (15 ml aliquot were removed as the negative control). Then 0.7 mM IPTG was added to the medium and the culture was continued at 300C.

The 15 ml samples were taken every hour from 3 hours through 7 hours. As indicated below in the Figure 12, the maximum volume of UvrA overexpression was found to be attained 6 hours after the induction of 0.7 mM IPTG.

Visual examination of the gel by Coomassie blue staining (Figure 12) reveals that UvrA protein production increased until a maximum level was reached at 6 hours post 0.7 mM IPTG induction. It inferred that a minimum culture time of 6 hours after induction will produce a substantial amount of the full-length UvrA_intein fusion protein. Longer culture time may not result in any significant increase in yield.

1 2 3 4 5 6 7 8 9 250 KDa

150

UvrA 100

Figure 12: Optimization of the time course for the overexpression of UvrA protein. Protein Marker (Lane 1). Protein UvrA (from pool- 32B) (Lane 2). Clarified crude extract from uninduced cells (Lane 3). Clarified crude extract from cells, induced at 30°C for 2 hours (Lane 4), 3 hours (Lane 5), 4 hours (Lane 6), 5 hours (Lane 7), 6 hours (Lane 8), 7 hours (Lane 9). 15 ml aliquots were removed at indicated intervals.

Small Scale of UvrA Purification by Chitin Beads to Check DTT Cleavage Efficiency

0.7 mM IPTG induction of UvrA_Intein fusion protein in 60 ml culture and 30 ml culture without IPTG were performed at 300C for 6 hours with shaking (200 rpm). The resulting 60 ml induced culture then was split into 2 of 30 ml tubes, and the cells from 3 sample tubes were harvested separately by centrifugation at 6,000 rpm for 10 minutes at 40C. Pellets were

28 resuspended in 1 ml lysis buffer for each sample with 1 mM PMSF and lysed by sonication (10 sec on, 10 sec off for 10 times). Clarified extracts (1 ml of each) were incubated with 50 µl chitin beads separately on a rotating rack for 2 hours. Recovered beads had 30 mM DTT added into one of the tubes that was from IPTG induction culture, and all 3 tubes of beads incubated at 40C overnight. All 3 samples from recovered beads and 3 supernatant tubes from the beads were analyzed by 10% polyacrylamide gel at 150 volts for 70 minutes and stained in Coomassie blue

(Figure 13). Comparing the samples from the recovered beads from the extracts of culture with both 0.7 mM IPTG and 30 mM DTT (Lane 4) to that from the extracts of culture with 0.7 mM

IPTG but without DTT (Lane 5), the cleavage of the protein UvrA from its intein tag in the sample with 30 mM DTT overnight incubation was most efficient as Figure 13 indicated.

Figure 13: DTT cleavage efficiency for the binding between fusion protein and chitin beads. Protein Marker (Lane 1). Protein UvrA (Lane 2). Recovered beads from the extracts of culture w/o IPTG induction (Lane 3). Recovered beads from the extracts of culture with 0.7 mM IPTG and 30mM DTT (Lane 4). Recovered beads from the extracts of culture with 0.7 mM IPTG but without DTT (Lane 5). Supernatant from sample of lane 3 (Lane 6). Supernatant from sample of lane 4 (Lane 7). Supernatant from sample of lane 5 (Lane 8). Small-scale UvrA_intein overexpression under 0.7 mM IPTG induction. 1 ml clarified crude extracts (from 30 ml of noninduced/induced cells) incubated with 50 ul of chitin beads at 4°C for 4 hours. Then, all the beads were recovered by centrifugation at 2,500xg for 20 seconds. 30 mM DTT was only added into one of the recovered beads tubes incubated with the extracts from induced culture. All 3 recovered beads tubes were incubated at 4°C overnight.

Cell Lysis Efficiency Comparison Between Sonication and French Press

Different methods were used for the preparation of cell lysates from E. coli cells: sonication and French press. Sonication is the most popular technique for lysing small quantities of E. coli cells such as 1 L of cell culture. Cells are lysed by liquid shear and cavitation. French presses are the alternative devices to lyse bacteria E. coli cells. Cells are lysed by a liquid shear created by pressuring the cell suspension and suddenly releasing the pressure.

As indicated in Figure 14A, after the sonication (10 cycles of 10 sec pulses with 10 sec pauses), most of the UvrA protein is still in the pellets compared to that in the clarified extracts.

After using the French Press (Figure 14B) for the cell lysis, the most of the UvrA protein shows in the clarified extracts instead of in the pellets.

The French press (model no. OMFA078A) was operated at 8,000 psi to achieve more adequate E. coli cells lysis compared to the sonication method which had much lower cell lysis efficiency in this case.

Pellets from A w/o IPTG 0.7 mM IPTG 250 bp UvrA-32B w/o IPTG 0.7 mM IPTG 150

100

Figure 14: Comparison of cells lysis effects between the methods of sonication and French press. (A) After the sonication, most of the UvrA protein is still in the pellets compared to the amount of the UvrA protein in the extracts. (B) After using the French press for the cell lysis, most of the UvrA protein shows in the extracts compared to the amount of UvrA protein in the pellets.

Purification of UvrA Protein from One Liter of Culture by T7 IMPACT System

For overexpression of UvrA protein from C41_pTYB2_UvrA E. coli cells, the fresh

AmpR transformants were inoculated into one liter of LB supplemented with ampicillin to a final

0 concentration of 100 µg/ml. The culture was first incubated at 37 C to an O.D600 of 0.6, and then the IPTG was added into it to a final concentration of 0.7 mM followed by induction at 300C for

6 hours. The cells were harvested, and the pellets were resuspended in 80 ml column buffer with

1 mM PMSF and lysed by one pass through French press at 8,000 psi. The clarified extracts were slowly loaded onto the chitin columns (10 ml) at a rate no faster than 0.5 ml/min. After the column was washed by 30 column volumes of column buffer, on-column cleavage reaction was induced by flushing the column with 3 column volumes of cleavage buffer with 30 mM DTT.

The flow in the column was stopped and left at 4 0C overnight. Protein UvrA was eluted by cleavage buffer without DTT and was collected in 1 milliliter fractions of each for a total of 20 milliliters.

As indicated in Figure 15, fraction 2 and fraction 4 (fractions are 1 ml each for 20 ml in total after cleavage with DTT and elution from the chitin column) were examined using PAGE for molecular weight and purity of isolated protein. Other samples including some major steps from the overproduction and purification of UvrA protein were also analyzed on a 10% SDS- polyacrylamide gel electrophoresis stained with Coomassie Blue and photographed. Such samples included those from cell extracts of noninduced cultures, 0.7 mM IPTG-induced cell cultures, the flow-through of extract loading, column washing, and on-column cleavage induction, and chitin resin, respectively. Fractions 1, 3,5,7,9 were also examined by 10% SDS- polyacrylamide gel electrophoresis stained with Coomassie blue for UvrA molecular weight and purity of isolated protein UvrA (Figure 16).

Fractions 1-10 and 11-20 were split into 2 dialysis tubings, pooled as pool-1 and pool-2

(as indicated in the Figure 17), and dialyzed against 1 liter of 50% glycerol storage buffer in the same beaker at 40C for 20 hours. The concentrations of protein UvrA of both samples from pool-

1 and pool-2 were measured by Pierce 660 nm Protein Assay, and they were 2.0 µM and 1.3 µM, respectively. The obtained purified UvrA protein from 1 liter of culture cells then was aliquoted into several of 1.5 ml microcentrifuge tubes and stored at -200C before its biological activity was confirmed.

1 2 3 4 5 6 7 8 9 10 250 KDa 150

100 UvrA 75

Figure 15: Expression and purification of the UvrA protein using the IMPACT system. Protein marker (Lane 1). Protein UvrA from pool- 32B (Lane 2). Clarified crude extract from uninduced cells (Lane 3). Clarified crude extract from cells, induced at 30°C for 6 hours (Lane 4). Chitin column flow through (F.T.) (Lane 5). Chitin column wash (Lane 6). Quick DTT wash to distribute DTT evenly throughout the chitin column (Lane 7). The supernatant (Lane 8) from chitin resin, to determine the cleavage efficiency. Fraction 2 and 4 of eluted UvrA after stopping column flow and inducing a self-cleavage reaction at 4°C overnight (Lane 9-10). Coomassie blue staining.

UvrA-32B Fraction-1 Fraction-3 Fraction-5 Fraction-7 Fraction-9 250 KDa

150

100

Figure 16: Comparison among the fractions of the eluted target protein UvrA. 1 ml fractions of each for 20 ml in total collection of eluted target protein UvrA.

UvrA-32B UvrA (Pool-1) UvrA (Pool-2) 250 KDa

150

100

Figure 17: Purified protein UvrA samples from pool-1 and pool-2. Purified UvrA protein, from one liter of culture cells, split into two dialysis tubings (fractions 1-10 as pool-1 and 11-20 as pool-2), was dialysed against the 50% glycerol storage buffer for 20 hours at 4 0C. The concentrations of protein UvrA from both samples of pool-1 and pool-2 were measured by Pierce 660 nm Protein Assay. The concentrations of the samples from pool-1 and pool-2 were 2.0 µM and 1.3 µM, respectively.

UvrABC Incision Assay to Characterize the Purified UvrA Protein in vitro

32P-labeled 58-bp duplex DNA substrate containing FABP [N-(20-deoxyguanosin-8-yl)-

4-ﬂuoro-4-aminobiphenyl] adducts was incubated with DNA excision nucleases UvrA, B, and C.

As indicated in Figure 18, the DNA incision products were present in the UvrABC DNA incision assay using the newly purified UvrA protein. In agreement with these incision data, the result supported our hypothesis that prokaryotic DNA excision nuclease UvrA protein can be purified by T7 IMPACT protein purification system.

Figure 18: UvrABC incision assay. DNA substrates, 32P-labeled at the 5’ termini, were incised by UvrABC in UvrABC buffer at 370C for a specified length of time. Aliquots were collected at 0, 10, 20, and 30 minutes from the reaction. The reaction was terminated by heating at 950C for 5 minutes. The products were denatured into single strands by addition of formamide loading buffer and heating to 950C for 5 minutes, and immediately plunged into ice to prevent reannealing. Digested (incision) products were then analyzed by gel electrophoresis on a 12% polyacrylamide sequencing gel under denaturing conditions with TBE buffer. 32P-labeled incision products or nonincision DNA bands in the gels were visualized using a Fuji FLA-5000 image scanner with MultiGauge V3.0 software.

CHAPTER 4

DISCUSSION

To express a target protein as a fusion protein to an affinity tag, such as GST

(Glutathione S-transferase), MBP (Maltose-Binding Protein), or His (histidine)-tag systems, is a commonly used technology for recombinant protein expression and purification (LaVallie and McCoy 1995). Some affinity tags even can help to elevate the expression level of the fusion protein and thus the yield of the target protein (LaVallie and McCoy 1995). This technology usually requires a separate protease to cleave the target protein from its affinity tag (Chong et al.

1998). The proteases treatment creates an extra step to the protein purification, and proteases are sometimes nonspecific and inefficient, which could become a potential limitation of this technology (Chong et al. 1998). We need good quality of UvrA, UvrB, and UvrC proteins for our research study in NER pathway. The prokaryotic DNA excision nuclease UvrA we currently are using in our lab was purified previously by 4-column steps methods including Q-sepharose column, DNA cellulose column, Mono-Q FPLC column, and G-200 FPLC column. These experimental procedures are very time-consuming and technically challenging. In this project

IMPACT was used as the cloning and protein purification system to purify a native recombinant

UvrA in a single-column (single-step). The protein splicing element intein of IMPACT is able to cleave a peptide bond without the use of a separate protease, and the intein-catalyzed peptide bond cleavage eliminates the need for a protease normally required by other affinity fusion systems.

To subclone UvrA DNA fragment, the C-terminus pTYB2 expression vector was chosen in the intein-tagged fusion protein construction with DH5α E. coli cells as the host organism. To

37 prepare for the UvrA DNA fragment to be cloned, PCR method was used for the amplification of

UvrA DNA fragments prior to the molecular cloning using pSST10_UvrA as the template. Both the cloning vector pTYB2 and UvrA DNA fragments were treated with 2 restriction endonucleases Nde I and Xho I, and these 2 restriction sites were used for the cloning of the amplified target gene of UvrA in fusion construction. These 2 cleavage sites are unique within the multicloning site (MCS) of vector pTYB2 and noncutters within UvrA fragment so that the vector can be cleaved only at a single site. To increase the recombination efficiency, the cleaved vector of pTYB2 was treated with an enzyme (alkaline phosphatase) that dephosphorylates the vector’s ends. The vector molecules with dephosphorylated ends are usually unable to self-ligate.

The DNA fragments prepared from the vector pTYB2 and UvrA gene from pSST10_UvrA plasmid were simply mixed together at 1:3 molar ratio and incubated with T4 DNA ligase to form 2 covalent phosphodiester bonds between 3’ hydroxyl end of one nucleotide with the 5’ phosphate end of another. The resulting DNA mixture that contained both correctly joined ends and randomly joined ends was sorted out by selectable marker and plasmid isolation after being transformed into the host organism DH5α E. coli cells. The vector pTYB2 has a gene that confers resistance to ampicillin. Cells harboring the pTYB2 will survive and grow when exposed to the ampicillin, while those that have failed to take up pTYB2 sequence will die. The desired products with vector pTYB2 covalently linked to UvrA DNA would be present, but undesired side products could be present also. To identify the organisms containing desired UvrA DNA inserts (recombinant DNA of pTYB2 and UvrA), it is necessary to examine a number of different clones (by restriction fragment analysis, polymerase chain reaction, and/or DNA sequencing) to be sure that the desired pTYB2_UvrA DNA construct was obtained. One clone

38 was selected and sequenced to confirm that the sequences at both junctions of the UvrA fragment insertion into the pTYB2 vector were correct.

Overexpression of the fusion protein from pTYB2 vector may be affected by the following factors: (A) E. coli strain; (B) cell culture conditions (cell density, temperature, etc.);

To achieve a maximal overexpression of fusion protein of UvrA_intein, C41 (DE3) E. coli strain derived from BL21 (DE3) (Miroux and Walker, 1996) was used. There is at least one uncharacterized mutation in the C41 strain. Compared to the BL21 (DE3) strain, C41 (DE3) cells are believed to increase the activity and/or amount of T7 RNA polymerase to have a higher expression level and prevent cell death associated with expression of many recombinant toxic proteins. The UvrA protein yield seems intermediate, and I may need to further optimize some other protein expression induction conditions, such as temperature, IPTG concentration, and buffer’s pH levels, etc.

The pTYB2_UvrA plasmid was transformed into C41 E. coli cells, and a small scale induced culture at 300C with 0.7 mM IPTG showed a maximum level of the fusion protein

UvrA_intein after 6 hours. In our lab purifications of the prokaryotic DNA excision nuclease

UvrB and UvrC were successfully performed previously using pTYB2 vector. Most of the cell culture and protein expression conditions are similar among these 3 kinds of proteins, except the

IPTG induction time course for UvrA, UvrB, and UvrC overexpression are different, and they are 6 hours, 2.5 hours, and 3 hours, respectively. The cell extract from 1 liter culture of

C41(DH3)_pTYB2_UvrA E. coli cells induced under 0.7 mM IPTG at 300C for 6 hours was applied onto the chitin column, and the cleavage of the induced fusion protein of UvrA_intein

39 was induced using cleavage buffer containing 30 mM DTT at 40C overnight. The eluted UvrA protein was dialyzed against the storage buffer with 50% glycerol at 40C and stored at -200C for a short time period and -800C for long-term storage. The UvrABC incision assay results showed that the UvrA protein purified by T7 IMPACT system is biologically active, which demonstrated that IMPACT system can be used for the purification of active UvrA protein in a single step. Our hypothesis for this project that prokaryotic DNA excision nuclease UvrA protein can be purified by T7 IMPACT system was supported by our experimental data.

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VITA

CATHY W. LIN

Education: Master of Science in Biology with a concentration in

Biomedical Sciences, 2011-2014, East Tennessee State

University, Johnson City, TN 37614

Chemistry Categorical Certificate Training Program, 2007-2008,

University of Maryland, School of Medicine, Baltimore, MD

21201

Bachelor of Medicine, 1984 -1990, Shanghai Medical School of

Fudan University, Shanghai, China

Professional Experience: Teaching Assistant, East Tennessee State University, Johnson

City, TN, 2011-2013

Andrologist /Jr. Embryologist, Medical Education Assistance

Corporation, Johnson City, TN, 2009-2010

Medical Laboratory Technologist, Patient First Clinical

Laboratory, Baltimore, Maryland, 2008-2009

Laboratory Technical Representative, Quest Diagnostics,

Auburn Hills, Michigan, 1998-2004

Medical Technologist in Hematology, Quest Diagnostics,

Auburn Hills, Michigan, 1994-1998, Wood Dale, Illinois, 1993-

1994

License: H (ASCP) cerified in Sept. of 1999

C (ASCP) cerified in Oct. of 2008

State of Tennessee Laboratory Technologist License